1. Field
This invention relates generally to the production of biologically active proteins from genetically engineered mammalian cell lines. Specifically, the invention is concerned with a novel expression vector containing an Epstein-Barr virus terminal repeat sequence which enhances integration of expression vectors into the genomic DNA in host mammalian cell lines.
2. Background
Many attempts have been made to increase the stable integration efficiency of expression vectors into genomic DNA by site specific integration.
Random, nonhomologous integration of input DNA into the host cell genome occurs more than 100 times more frequently than targeted homologous recombination (Thomas et al., 1987, Cell 51: 503-512). However, homologous recombination using hotspot, e.g. hypervariable minisatellite DNA, was shown to occur more frequently than random recombination between two defective plasmids in mammalian cells (Wahls et al., 1990, Cell 60: 95-103).
Autoantigenic cellular protein was isolated by Sun et al. (1994, Proc Natl Acad Sci USA 91: 8646-8650). This protein was identified as terminal repeat binding protein, or TRBP. Two terminal repeat binding sites (TRBS1 and TRBS2) for terminal repeat binding protein were also identified by Sun et al. They observed that TRBP binds sequences present in repetitive cellular DNA, e.g. variable-number tandem repeats (VNTR) and immunoglobulin heavy chain class switch regions.
The terminal repeat binding protein binds to G-rich regions of terminal repeats of Epstein-Barr virus (EBV-TR). EBV-TR takes part in processing and packaging of virion DNA (Zimmermann et al., 1995, J Virol 69: 3147-3155). The EBV-TRs are involved in the integration into chromosomal DNA (Matsuo et al., 1984, Science 226: 1322-1325) and in the circularization event of the genome after infection. These sequences are the essential elements for cleavage and packaging of the EBV virion DNA (Hammerschmidt et al., 1989, Nature (London) 340: 393-397; Zimmermann et al. J Virol, 1995, 69: 3147-3155). These data indicate the important role of the EBV-TR sequence in the recombination events. Therefore, we tested EBV-TR for integration events in deriving clones from the transfected cells.